While tissue engineering offers the promise of improved clinical treatment for numerous disorders, obstacles that have limited the translation of laboratory successes into clinical practice must be overcome before this potential is realized. This holds true for the tissue engineered heart valve (TEHV), a living, growing valve substitute that would significantly improve the treatment of congenital heart disorders in children and degenerative valve disorders in adults. However, scientific and regulatory challenges limit the practicality and attractiveness of previous heart valve tissue engineering strategies. The use of complex bioreactors systems that are not disposable or patient-specific introduces a substantial regulatory obstacle that must be overcome. We have addressed this issue through the development of a fully disposable, single-use bioreactor for heart valve seeding and conditioning, permitting the investigation of clinically feasible TEHV processing strategies. Scaffold safety is also of major concern, as the implanted construct must be fully functional as a valve from the time of implantation forward. This concern can be mitigated through the use of decellularized heart valve scaffolds, which have a record of clinical safety; however, previous difficulties in introducing sub-surface cells into the leaflet of the decellularized heart valve hae dampened enthusiasm for this particular TEHV scaffold. Using our custom bioreactor, we have overcome this limitation, successfully creating a pilot population of bone marrow mononuclear cells (BM-MNCs) within the leaflet. The objectives of this project are to determine the potential for ex vivo maturation of this pilot BM-MNC population through extended bioreactor processing and to evaluate the subsequent in vitro inflammatory response to the seeded tissue. Two Specific Aims have been formulated to accomplish these objectives. In the first Aim, extended bioreactor processing will be utilized to determine the extent to which the pilot BM-MNC population proliferates and differentiates during the culture period. We will also evaluate the potential for remodeling of the decellularized scaffold. Aim 2 will utilize an in vitro macrophage cytokine signaling assay to assess the inflammatory response to the seeded tissue. Accomplishing these two Specific Aims will elucidate the recellularization mechanisms of the decellularized heart valve in the context of heart valve tissue engineering.